Bottom Line:
However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences.We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences.Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

Background: The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized.

Principal findings: Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences.

Conclusions/significance: Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

pone-0062799-g002: Taxonomic assignments of rRNA gene reads from the Neanderthal and the cave sediment samples.Bacterial community composition patterns at (A) the phylum level and (B) within Actinobacteria. The Neanderthal rRNA gene sequence reads were classified based on BLASTn searches to the SSURef111NR database in SILVA, modified as described in the methods section. The cave sediment rRNA gene sequence reads were classified based on a maximum likelihood phylogeny that included related reference sequences. Bacterial composition patterns at the phylum level were inferred from PCR-amplifications of the bacterial DNA extracted from the cave sediment using (A) universal primers and (B) actinobacterial-specific primers. Scales refer to % of all (A) Bacteria and (B) Actinobacteria.

Mentions:
We used the modified rRNA database search approach outlined above for the taxonomic analyses of the fossil metagenome data sets. Thus, broad taxonomic assignments of both the untreated and the treated Neanderthal data sets were inferred from BLASTn searches (E<10−10) against the eSILVA rRNA gene sequence database. Using this procedure, we identified from 48,000 to 140,000 rRNA gene reads per sample, of which 89% to 96% were classified as Bacteria, less than 10% as Eukaryota and less than 2% as Archaea (Table S5). Within bacteria, the diversity at the phylum level was low, with a large majority of the bacterial sequence reads, 74%–95%, being assigned to the Actinobacteria (Figure 2A, Table S6). Proteobacteria accounted for another 3%–14% and represented the second most abundant group (Table S6). Finally, within the Actinobacteria, the majority of sequence reads, 20%–35% were assigned to the Streptomycetales (Figure 2B, Table S7). There was no significant difference (t-test in R, p-value >0.32) in the content of Streptomycetales between two different extractions from the bone (Figure S2A, Table S8). Nor was there any difference in the abundance of Streptomycetales between subsets of short (<150 bp) and long (>150 bp) sequence reads (Figure S2B).

pone-0062799-g002: Taxonomic assignments of rRNA gene reads from the Neanderthal and the cave sediment samples.Bacterial community composition patterns at (A) the phylum level and (B) within Actinobacteria. The Neanderthal rRNA gene sequence reads were classified based on BLASTn searches to the SSURef111NR database in SILVA, modified as described in the methods section. The cave sediment rRNA gene sequence reads were classified based on a maximum likelihood phylogeny that included related reference sequences. Bacterial composition patterns at the phylum level were inferred from PCR-amplifications of the bacterial DNA extracted from the cave sediment using (A) universal primers and (B) actinobacterial-specific primers. Scales refer to % of all (A) Bacteria and (B) Actinobacteria.

Mentions:
We used the modified rRNA database search approach outlined above for the taxonomic analyses of the fossil metagenome data sets. Thus, broad taxonomic assignments of both the untreated and the treated Neanderthal data sets were inferred from BLASTn searches (E<10−10) against the eSILVA rRNA gene sequence database. Using this procedure, we identified from 48,000 to 140,000 rRNA gene reads per sample, of which 89% to 96% were classified as Bacteria, less than 10% as Eukaryota and less than 2% as Archaea (Table S5). Within bacteria, the diversity at the phylum level was low, with a large majority of the bacterial sequence reads, 74%–95%, being assigned to the Actinobacteria (Figure 2A, Table S6). Proteobacteria accounted for another 3%–14% and represented the second most abundant group (Table S6). Finally, within the Actinobacteria, the majority of sequence reads, 20%–35% were assigned to the Streptomycetales (Figure 2B, Table S7). There was no significant difference (t-test in R, p-value >0.32) in the content of Streptomycetales between two different extractions from the bone (Figure S2A, Table S8). Nor was there any difference in the abundance of Streptomycetales between subsets of short (<150 bp) and long (>150 bp) sequence reads (Figure S2B).

Bottom Line:
However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences.We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences.Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

Background: The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized.

Principal findings: Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences.

Conclusions/significance: Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.